Chlamydiae are obligate parasitic bacteria that divide and multiply within the cytoplasm of eucaryotic host cells. Infection by chlamydiae can result in infertility, ectopic pregnancy, acute epididymitis, proctitis and blindness if left untreated. The long-term goal of this project is to identify, characterize, and sequence new genes in the chlamydial genome that are necessary for its infectivity and intracellular pathogenesis. The proteins coded for by these genes will be identified and characterized as to function to determine if these proteins can serve as new potential targets for chemotherapy and or immunotherapy. The specific aim of this project is two-fold. First, cloning techniques will be used to identify possible topoisomerase genes that may be part of the chlamydial genome and that may be necessary for intracellular replication of chlamydiae after infection and may be necessary for production of infectious elementary bodies (EBs) during the developmental cycle. Both a plasmid and cosmid library of chlamydial DNA will be screened by complementarity and/or hybridization techniques with appropriate probes to try and identify clones containing either type I or type II topoisomerase genes. If such a gene is found, it will be cloned in an appropriate vector in order to purify the protein and to study its enzymatic properties. Derivatives of camptothecin and other type I topo inhibitors will be tested as potential drugs against chlamydial infection. Secondly, purified chlamydial EB particles and infected HeLa cells will be assayed for a 3'nucleotidase enzyme activity to determine if chlamydiae produce this enzyme as a critical component of the metabolic pathways required for their replication. A 3'-nucleotidase enzyme activity has been identified and purified in Leishmania donovani promastigotes as a surface membrane component. This parasitic protozoan grows within lysosomes of macrophages, differentiates into nonmotile amastigotes, and multiplies intracellularly producing cutaneous, mucocutaneous, or visceral leishmaniasis. Chlamydiae also divide and multiply in a similar subcellular location within a variety of eucaryotic cells and may also require similar enzymes to carry out their parasitic metabolic functions. If this enzyme activity or other similar nucleotidase activities can be identified, the enzyme will be purified and the gene for the enzyme will be cloned from a chlamydial DNA library based on microsequencing data obtained from the purified enzyme. If this enzyme activity is critical for replication of the chlamydiae, specific inhibitors of this enzyme will be identified and/or synthesized. Since 3'-nucleotidase activity has not been identified in eucaryotic cells, this enzyme would be an attractive candidate as a new chemotherapeutic target. If this enzyme is located on the surface membrane of chlamydial EBs, it may also serve as an antigenic target for a potential vaccine.